The object of the invention is a ready-to-use culture medium for microorganisms, comprising nutrients and hydrogel based on cellulose ethers. The culture medium may be dried on a solid support, stored dry and moistened when taken into use, either directly with a sample to be tested or with water. Culture medium device may be immersed in a sample solution to be tested, or it may be pressed against a damp sample surface to be tested, after which the medium is incubated at a suitable temperature until microorganisms have grown to visually recognizable colonies.
The determination of the amount and species of the microorganisms which cause infections and contamination is important when either a suitable method of treatment in medication or a defensive method in food industry and elsewhere in the environmental hygiene is chosen. Several culture media and culture media devices for detecting various bacteria and fungi have been developed for this purpose.
In the microbiology that diagnoses diseases the aim is to determine and identify the microorganisms which cause infections, i.a. bacteria (both aerobic and anaerobic bacteria) and fungi (yeasts and molds), as exactly as possible from various samples of human and animal origin (e.g. urine, blood, serum, plasma, cerebral spinal fluid, pleural fluid, ascites fluid, pus, wound secretion, sputum, stool, and pharyngeal specimen) so as to be able to choose an effective, curative method of treatment. Often also the susceptibility of microbes to various antimicrobial drugs is to be determined. Various culture media and culture media devices are used for this purpose.
Microorganisms cause problems also in several different fields of industry. If they succeed in accumulating in processes, the health hazards and contamination problems caused by them will become difficult to handle. Usually microbes can effectively resist cleaning inside a so called biofilm layer composed of the solid material of microbe cells and process liquids, which layer gives protection against the effect of disinfectants and antiseptic agents, as well as against the effect of antibiotics, and inside which microbes may survive even for long periods of time.
Several production processes require a high hygienic level, because even a slight growth of microorganisms may ruin the whole product. Lowered hygiene causes problems especially in food industry, in health care, in medical treatment and in water systems. Other kinds of problems, such as slime formation caused by bacteria in the vessels and pipe systems of process industry, may occur especially in wood processing industry, and molds may appear everywhere in ventilation pipes. In industry it has been found that microbes contribute also to other problems, such as at the emergence of corrosion. Microbes and the problems caused by them are found also elsewhere, such as on the tiles of bathroom, on sauna benches and in swimming pools. In hospitals pathogenic microbes may e.g. colonize in a ventilation system and thus cause hospital infections. Therefore a continuous follow-up of hygiene by determining the amounts of microbes is in use in most facilities where the level of hygiene is to be realized.
The classical methods for culturing microorganisms are based on culture media where the gelling agent is agar-agar isolated from algae. Culture media have to be either prepared laboriously from a dry powder or bought in a ready-to-use form. Commercially available ready-to-use culture media are ready in swollen, wetted form. The storage time of self-made and of industrial, moist culture media which contain agar-agar is limited, because they dry easily and cannot be moistened again. In order to maintain moisture and sterility for as long as possible, this kind of culture media have to be stored in very tight packagings which need plenty of room.
In the culture medium of the U.S. Pat. No. 3,046,201, the gelling agents used instead of agar-agar include cross-linked polyacrylamide hydrogels or mixtures of polyacrylamide and gelatine, silica gel or starch. The gel is not dried and it does not have the property of absorbing sample liquid.
In the method according to the U.S. Pat. No. 3,360,440, a dry gel is prepared by mixing nutrients with cellulose ether and by lyophilizing the mixture. Before lyophilization, microorganisms may be added to the mixture, and thus a ready-to-use, preservable culture is obtained. The dry gel is resuspended with sterile water before it is used for the determination of microbes or, if microbes have been added before lyophilization, before growing of microbes. In microbe assays, the microbes of the sample are absorbed within the gel and grow thus differently as to their morphology compared to the growth on usual media where growth takes place on the surface of the gel. This considerably complicates the readability of the results and thus reduces the reliability of the assays. Further, lyophilization is a multistage process and requires relatively expensive equipment.
In the test kit according to said U.S. Pat. No. 3,360,440, water and the dried gel have been included in different parts of a reservoir. After resuspension the gel may be transferred on a desired support by pushing the reservoir wall. The test kit is complicated and is not ready-to-use.
GB 1 295 337 discloses a dehydrated hydrogel which is integrally bonded to a culturing container by means of radiation and which comprises e.g. crosslinked poly(ethylene oxide), polyvinyl pyrrolidone and polyvinyl alcohol. Nutrients are added to the gel immediately after its preparation or just prior to use.
In addition to this, methods are known where nutrients and gel are absorbed by an absorbing membrane, which may be a filter paper or other equivalent, absorbing support layer. The gel may also be spread on the surface of a membrane, or the gel is replaced by a membrane which is impermeable to bacteria. In these methods the absorbing membrane maintains inside itself the moisture needed for growth. Methods like this are disclosed in the patent publications U.S. Pat. No. 3,881,993, EP 0 374 905, U.S. Pat. No. 3,814,670 and EP 0 006 192, which are examined in more detail in the following.
In the method of the U.S. Pat. No. 3,881,993, nutrients, reagents and a gelling agent have been absorbed by an absorbing layer, which acts also as a supporting structure, and the medium has been dried under negative or positive pressure. As a gelling agent inert gum, linear polysaccharides or sodium alginate are used. In the method according to EP 0 374 905, gelling agents are absorbed by a filter paper. Sodium alginate is used as a gelling agent. The test medium according to the method is used wet. The method according to the U.S. Pat. No. 3,814,670 is similar to the above ones, with the exception that the gel layer is spread on the surface of an absorbing membrane. As a gelling agent agar, gelatine, cellulose gums, carrageens, alginates, albumins, polysaccharides or polypeptides may be used. The culture medium has been dried. EP 0 006 192 discloses a dry culture medium where a culture medium cartoon is covered by a plastic membrane, which is a homopolymer of vinylacetate or acrylic acid esters, a copolymer of vinylpropionate and vinylacetate, vinylpropionate and vinylchloride, vinylacetate and maleic acid esters, acrylnitrile, acrylic acid esters and vinylpropionate or butadiene and styrene. As an opening agent polyethyleneglycols, polyethyleneoxides, polyvinylpyrrolidone, polyvinylalcohols, partly saponified polyvinylesters, mixed polymers of vinylpyrrolidone and vinylesters, or cellulose derivatives, such as hydroxyalkyl cellulose, are used.
In the U.S. Pat. No. 5,494,823, a mixture comprising nutrients and a gelation agent is coated with an absorbent fibrous sheet into which the sample solution is dispersed by the phenomenon of capillary attraction. After the mixture containing the gelation agent has absorbed moisture and formed a gel, the fibers of the absorbent fibrous sheet are buried and adhered into the mixture.
JP 94-172178 discloses a cell culture carrier comprising two hydrogels, the other gel containing e.g. particles microbeads, fibre, sponge, etc. and functioning as a supporting structure.
A disadvantage with above mentioned methods is that the gelling agent of culture medium needs supporting layers to remain consistent, which makes even the preparation of media difficult. Those methods wherein the gelling agent is also absorbed by an absorbing layer further have the disadvantage that the surface on which microbes grow is rough, and thus to an observer the microbes look totally different from those growing on the surface of agar in a classical detection method.
Also known are culture media where gelling agents are powders which are soluble in cold water. They are attached on a hydrophobic support with the help of an adhesive layer, like in the U.S. Pat. No. 5,089,413 and U.S. Pat. No. 4,565,783, as well as in the Wo publication 93/12218. The culture medium often further comprises an air-permeable membrane. A hydrophobic support may be directly covered with the gel, whereby the gel has been crosslinked with ionic bonds by adding salts into the gel. Culture media like this have been used in the methods according to the U.S. Pat. No. 5,089,413 and 4,565,783 only as streakable media. However, a disadvantage is that when crosslinking is carried out with the help of salts, the growth of microbes is often inhibited.
In the methods according to the patents U.S. Pat. No. 5,089,413 and U.S. Pat. No. 4,565,783, copolymers of isooctylacrylate and acrylamide or acrylic acid, or copolymers of 2-methylbutylacrylate and acrylic acid or silicone gum are used as adhesives. As gelling powder, alginine, carboxymethyl cellulose, hydroxyethyl cellulose, guar gum, locust bean gum, xanthan gum or mixtures of these are used. In the method according to WO 93/12218, a copolymer of isooctylacrylate and acrylamide or N-vinylpyrrolidone is used as an adhesive layer. To this adhesive layer is attached a cold-water-soluble powder, which is a mixture of super absorbent and gelling agents. The gelling agent may be alginine, guar gum, hydroxymethyl cellulose, locust bean gum, carboxymethyl cellulose, xanthan gum or a mixture of these. The superabsorbent agent is a glycol modified polysaccharide, a graft polymer of starch and sodiumacrylate acrylamide or a mixture of these. Except of the streakable medium, in these methods microbes grow inside the gel. A disadvantage is therefore that the morphology of colonies is not similar to the morphology on agar-agar surfaces.